Chromatographic methods generally are used to separate and/or purify molecules of interest such as proteins, nucleic acids and polysaccharides from a mixture. Affinity chromatography specifically involves passing the mixture over a matrix having a ligand specific (i.e. a specific binding partner) for the molecule of interest bound to it. Upon contacting the ligand, the molecule of interest is bound to the matrix and thus retained from the mixture. Affinity chromatography provides certain advantages over other types of chromatography. It provides a purification method that is highly specific, fast, economical and high yielding.
In one application affinity chromatography may be used to purify proteins such as monoclonal antibodies. As an example, antibodies of the IgG subtype may be affinity purified by passing them over a matrix having protein A or protein G bound to it (Boyle and Reis, 1987, Biotechnology 5:697; Hermanson et al., 1992, Immobilized Affinity Ligand Techniques, Academic Press; U.S. Patent Publication No. 2006/0134805).
Methods of attaching protein ligands, such as protein A and protein G to a solid support, e.g., chromatographic media have been previously described, see, e.g., Hermanson et al. 1992, Immobilized Affinity Ligand Techniques, Academic Press; U.S. Pat. Nos. 5,874,165; 3,932,557; 4,772,635; 4,210,723; 5,250,6123; European Patent Application EP 1 352 957 A1, WO 2004/074471. Typically, the media is activated with a reactive functional group (“activated group”) such as an epoxide (epichlorohydrin), cyanogens (cyanogens bromide (CNBr)), N,N-disuccinimidylcarbonate (DSC), aldehyde or an activated carboxylic acid (e.g. N-hydroxysuccinimide (NHS) esters, carbonyldiimidazole (CDI) activated esters). These activated groups can be attached directly to the base matrix, as is the case for CNBr, or they can be part of a “linker” or spacer molecule which is typically a linear chain of carbon, oxygen and nitrogen atoms, such as the ten membered chain of carbon and oxygen found in the linker butanediol digycidyl ether (a common epoxide coupling agent). The activated media is then equilibrated with the protein ligand under coupling conditions. Once the coupling reaction is finished, the media is washed thoroughly. For Protein A, typically 4-6 mg of protein (ligand density) may be loaded per ml of media resulting in an IgG maximum static capacity (Qs) of 40 g/L. Protein ligand density determines static capacity. The static capacity provides an upper limit of the protein capacity that can be used during the chromatographic separation. In general, the dynamic or loading capacity (Qd) correlates the static capacity with for a given media.
A recombinant form of protein A suitable for attachment to an agarose support has also been described. The recombinant form of the protein is engineered to have a terminal cysteine, see, e.g., U.S. Pat. No. 6,399,750; GE Healthcare Product Literature for r-Protein A Sepharose Fast Flow, Mabselect® and Mabselect Xtra®. Using this recombinant Protein A, IgG static capacities ranging from 55-70 g/L have been achieved. However, one limitation associated with using this recombinant protein A ligand is that it must be genetically engineered to contain the selective coupling functionality which may be time consuming and expensive.
Another strategy for coupling of a protein ligand to a solid support is “linker assisted coupling”. Linker assisted coupling, in contrast to ligand assisted coupling described in detail below, is characterized by its reliance on a single molecule which includes both the ligand (i.e. the specific binding partner of the target molecule to be purified) and a suitable associative functionality which may assist in coupling the ligand to the solid support. This technique involves engineering a functional group (such as a charged amine) into to a linker or spacer designed to couple the protein ligand of interest. The linker in these systems also possesses an activated group for coupling the protein to the linker. The linker is first associated with the solid support and then contacted with protein. Therefore, the single molecule linker serves the dual role of covalently attaching the protein to the matrix and interacting non-covalently with the protein. This provides some pre-association of the entities before/during the coupling reaction. This technique was exploited to couple glycans to a membrane surface for membrane blotting of electrophoresis gels (U.S. Pat. No. 5,543,054; Charkoudian et al., 1995, Analytical Letters, 28:1055).
Another example of linker assisted coupling is found in the commercial product Affi-Gel 15® (BioRad, Hercules, Calif.). Affi-Gel 15® is an agarose support derivatized with an NHS activated carboxylic acid as part of a linker arm containing a positively charged functionality. The positively charged functionality is a secondary amine. This amine is protonated at pH 7.4 and allows for coupling of proteins with an isoelectric point (pI) less than 6. The properties of the Affigel matrix, and other such ligands created by linker assisted coupling have limitations because the associative group (positive charge) is part of the protein coupling linker affording a fixed 1:1 ratio of the two functionalities. Another charged linker arm is described for coupling Protein A to agarose in U.S. Pat. No. 5,260,373. Here a shorter linker arm comprised of arginine is used to facilitate protein coupling to an agarose support. The arginine linker is activated with NHS and carries a positive charge. The results suggest, however, that only a modest improvement in IgG binding capacity is attained compared to non-linker assisted coupling. Moreover, the 1:1 ratio described above is maintained here as well. Protein A bound to a solid support via a linker has recently been described in U.S. patent application Ser. No. 10/928,731.
Scale up of fermentation and tissue culture methods, e.g., for the production of monoclonal antibodies, has resulted in both increased volumes and increased concentrations of target molecules produced by these methods. Product concentrations exceeding 1 gram per liter are not uncommon. These products require purification before they may be marketed. Accordingly, a need exists for a chromatographic matrix having enhanced binding capacity that provides good product yield, and which is economical and easy to manufacture. Various embodiments of the invention described below meet these and other needs.